Chemical cross-linkers are compounds which have the ability to covalently link two or more molecules with each other. Cross-linkers are used, e.g., to link protein molecules to one another, proteins to nucleic acids, carbohydrates to proteins or carbohydrates to other carbohydrates. The agents can also be used for the covalent attachment of proteins, nucleic acids or other small molecules to solid supports for their immobilization. These chemical cross-linker compounds have generally been symmetrically bifunctional in nature and can be further subdivided into three major classes: (1) homobifunctional reactive cross-linkers which contain the same reactive group on both ends of the molecule (X-X); (2) heterobifunctional reactive cross-linkers which have different reactive groups in their structure (X-Y), the reactive groups X and Y usually having different chemical specificities toward functional group(s) on the molecule(s) to be cross-linked; and (3) cleavable cross-linkers which contain in their structure one or more functional groups which can be cleaved under controlled conditions and which can either be homo- or heterobifunctional in nature.
A wide range of chemical cross-linkers have heretofore been developed. Representative homobifunctional cross-linkers (X-X) include bisactivated esters, bisimidates, alkylating agents, diazides, dialdehydes, triazines and benzoquinone. Heterobifunctional cross-linkers (X-Y) employed to date generally incorporate in their structures a combination of the reactive groups mentioned above. For example, combinations of acylating and alkylating groups or acylating and photoactivatable functionalities have been employed in this class of cross-linkers. Cleavable cross-linkers (either homo- or heterobifunctional) have metastable bonds that can be cleaved under controlled conditions, such as by using mild chemical treatment. The most widely employed metastable functionalities in cross-linkers are disulfides (which can be cleaved in the presence of free sulfhydryls) and vicinal hydroxyls (which can be cleaved by sodium periodate oxidation under mild conditions).
Cross-linking agents have a wide range of chemical and biochemical applications. These can be broadly classified into three categories: (1) cross-linking soluble compounds; (2) attaching soluble compounds to solid surfaces; and (3) cross-linking synthetic polymers (such as used in chromatographic methods) in order to improve their mechanical strength.
Cross-linkers have been extensively used to study the quaternary structure of multi-subunit proteins and to investigate spatial relationships of subunits and their interaction within the proteins. Both homo- and heterobifunctional cross-linkers have also found widespread applications in the preparation and use of immunodiagnostic reagents. For example, in enzyme linked immunosorbant assays (ELISA), the enzyme label is covalently linked through the use of a cross-linker to a hapten, antigen or antibody. Similarly, nonenzymatic labels can also be attached using such cross-linkers. Conjugation of a toxin to a specific antibody in the preparation of immunotoxin can be achieved by using such reagents. Attachment of nonimmunogenic polymers, such as poly(ethylene glycol), to a protein via bifunctional cross-linkers may result in a conjugate molecule that can evoke immune tolerance.
Using the same principles of cross-linking, it is possible to covalently attach proteins, nucleic acids or other ligands to a solid surface using cross-linking agents. For example, a solid surface containing a suitable functional group can attach itself to one end of a cross-linker. The other end of the cross-linker can then react with a functional group on the protein which can thus covalently attach itself to the solid support.
Many of the known cross-linking reagents have serious disadvantages, limiting their practical use. For a cross-linking reaction to be complete, the reagent should have a reasonable half-life in the buffer system or other medium used; in other words, the rate of hydrolysis of the reagent must be sufficiently slow that the reagent is available for the period of time necessary for the cross-linking reaction to be substantially completed. However, this is not the case with respect to certain reagents; for example, the half-life of bisimidates is about 4 minutes under acidic or slightly alkaline conditions (pH 8.0). In general, cross-linking reactions are carried out at a pH of 8.5; consequently, one must either keep adding reagent to compensate for losses due to hydrolysis until the desired cross-linking reaction is complete, or use a much higher pH with addition of reagent during reaction to facilitate cross-linking (i.e., pH 10.0 or above). Most proteins and nucleic acids are not stable at these pH levels. Moreover, some of the cross-linkers described above also suffer from problems of poor solubility in the types of solvent which would most commonly be employed with the materials it is desired to cross link.
2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) has heretofore been used in the activation of hydroxyl functions on solid supports, generally polymeric in nature and containing a plurality of hydroxyl groups (Ngo, T. T., "Facile Activation of Sepharose Hydroxyl Groups by 2-Fluoro-1-Methylpyridinium Toluene-4-Sulfonate: Preparation of Affinity and Covalent Chromatography Matrices," Biotechnology 4: 134 (1986)). The thus-activated support contains 2-alkoxy-1-methylpyridinium groups bound to its surface. This chemical entity can easily be displaced as 1-methyl-2-pyridone by nucleophiles (such as primary amino or sulfhydryl groups) from another molecule, thereby resulting in the covalent attachment of the nucleophile to the solid support.
Thus, U.S. Pat. No. 4,582,875 describes a method for preparing a stable and hydrolysis-resistant coupling product of a polymeric gel and an organic ligand, in which the organic ligand is covalently bonded directly to a carbon atom in the polymeric gel. According to this patent, a reactive derivative of a polymeric hydroxyl-containing gel is formed by reacting the gel with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) and then reacting the activated carrier with a ligand containing a primary amino acid or sulfhydryl group. The patent is directed to activation of hydroxyl groups of polymeric substances containing at least one hydroxyl group; specific examples of such polymeric substrates include polysaccharides, poly(ethylene glycol), poly(vinyl alcohol) and poly(hydroxyethyl methyl acrylate).
While according to U.S. Pat. No. 4,582,875 the contemplated carriers may be either water soluble or water insoluble, they are clearly polymeric in nature. The activated polymeric carriers of this patent would thus not be suitable for use as cross-linking agents, where structural or configurational limitations imposed by the materials to be cross-linked would effectively preclude the use of such high molecular weight materials.
It is an object of the present invention to provide novel, relatively low molecular weight cross-linking agents which are suitably soluble in solvents of interest in the treatment of materials of biological and chemical interest and have reasonable half-lives in solutions varying over a wide range of pH values as commonly employed in analytical and preparative chemical methods.
It is a further object of the present invention to provide a method for the preparation of bi- and polyfunctional cross-linking agents as described above in high yield and of reasonable purity.
It is yet another object of the present invention to provide methods for covalently bonding two or more organic molecules with each other using the novel cross-linkers of the invention.